The ATP-binding cassette transporter associated with antigen processing (TAP) plays a key role in the adaptive immune defense against infected or malignantly transformed cells by translocating proteasomal degradation products into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. The broad substrate spectrum of TAP, rendering peptides from 8 to 40 residues, including even branched or modified molecules, suggests an unforeseen structural flexibility of the substrate-binding pocket. Here we used EPR spectroscopy to reveal conformational details of the bound peptides. Side-chain dynamics and environmental polarity were derived from covalently attached 2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probes, whereas 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled peptides were used to detect backbone properties. Dependent on the spin probe's position, striking differences in affinity, dynamics, and polarity were found. The side-chains' mobility was strongly restricted at the ends of the peptide, whereas the central region was flexible, suggesting a central peptide bulge. In the end, double electron electron resonance allowed the determination of intrapeptide distances in doubly labeled peptides bound to TAP. Simulations based on a rotamer library led to the conclusion that peptides bind to TAP in an extended kinked structure, analogous to those bound to MHC class I proteins.adaptive immune system | antigenic peptide binding | peptide conformation | double electron electron resonance EPR | site-directed spin labeling

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotidebinding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.The transporter associated with antigen processing (TAP) 4 plays a prominent role in the antigen-processing pathway via major histocompatibility complex (MHC) class I molecules (1, 2). A fraction of proteasomal degradation products is translocated into the endoplasmic reticulum by the TAP complex and loaded onto MHC class I molecules. Peptide-loaded MHC complexes can pass the endoplasmic reticulum quality control and traffic to the cell surface, where they display their antigenic cargo to cytotoxic T-lymphocytes, which can thereby efficiently recognize and eliminate infected or malignantly transformed cells. TAP belongs to the superfamily of ATP-binding cassette (ABC) transporters, which translocate a very broad spectrum of substrates across membranes (3, 4).The TAP complex forms a TAP1/TAP2 heterodimer; each subunit contains a transmembrane domain (TMD) followed by a cytosolic nucleotide-binding domain (NBD). The translocation mechanism can be dissected into an ATP-independent peptide binding and an ATP-dependent translocation step (5). TAP1 and TAP2 are required and sufficient for both processes (5, 6). Peptide binding is composed of a fast association step followed by slow structural reorganization of the transport complex (7,8). Previous studies demonstrated that peptide binding to the TMDs triggers ATP hydrolysis by the NBDs (9, 10). TAP preferentially binds peptides with a length of 8 -16 amino acids (5). Using combinatorial peptide libraries, the first three N-terminal and the last C-terminal residues were identified as critical for peptide binding, whereas amino acids between these "anchor" positions do not significantly contribute to the substrate recognition (11). Remarkably, TAP can also bind peptides with large bulky side chains, including cross-linkers, fluorophors, or extended side chains (9, 12, 13). Strikingly, however, sterically restricted peptides that bind but are not transported do not stim...

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an ␣-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15 N and 2 H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is ϳ15°(؎7°). The alignment of both structured domains exhibits a mosaic spread of ϳ10°. A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.Survival of vertebrates is strongly dependent on the adaptive immune system, which confers protection against pathogens or cancer. On the cellular level, the major histocompatibility complex (MHC) 4 class I-dependent pathway of antigen processing allows for presentation of antigenic peptides at the cell surface and can trigger the elimination of virus-infected or malignantly transformed cells by cytotoxic T lymphocytes (1-3). The efficiency of antigen presentation depends on the transporter associated with antigen processing (TAP), a member of the ATPbinding cassette protein family that translocates peptides generated by proteasomal protein degradation into the endoplasmic reticulum for loading onto MHC class I molecules (4). This process requires a macromolecular peptide-loading complex of ϳ1 MDa comprising the transporter subunits TAP1 and TAP2, the MHC class I heavy chain, the non-covalently associated ␤ 2 -microglobulin, the adaptor protein tapasin, and several auxiliary factors (5-9).Herpes simplex virus type 1 is a highly abundant human pathogen that achieves lifelong persistence in the ganglia of the nervous system. Upon exogenous stimuli, it can be repeatedly reactivated and infect related mucosal tissues leading to clinical symptoms (10). To escape immune surveillance, herpes simplex virus compromises the host's cytotoxic T lymphocyte response via ICP47, an 88-amino-acid immediate early gene product (IE12) that blocks TAP function (11-15). In the absence of a functional TAP transporter (within 3 h of infection with herpes simplex virus), peptide loading onto MHC class I molecules is inhibited, and as a consequence, empty MHC I molecules are retained in the endoplasmic reticulum and ultimately directed to proteasomal degradation. By binding with nanomolar affinity t...

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.

In the human genome, the five adenosine triphosphate (ATP)-binding cassette (ABC) half transporters ABCB2 (TAP1), ABCB3 (TAP2), ABCB9 (TAP-like), and in part, also ABCB8 and ABCB10 are closely related with regard to their structural and functional properties. Although targeted to different cellular compartments such as the endoplasmic reticulum (ER), lysosomes, and mitochondria, they are involved in intracellular peptide trafficking across membranes. The transporter associated with antigen processing (TAP1 and TAP2) constitute a key machinery in the major histocompatibility complex (MHC) class I-mediated cellular immune defense against infected or malignantly transformed cells. TAP translocates the cellular "peptidome" derived primarily from cytosolic proteasomal degradation into the ER lumen for presentation by MHC class I molecules. The homodimeric ABCB9 (TAP-like) complex located in lysosomal compartments shares structural and functional similarities to TAP; however, its biological role seems to be different from the MHC I antigen processing. ABCB8 and ABCB10 are targeted to the inner mitochondrial membrane. MDL1, the yeast homologue of ABCB10, is involved in the export of peptides derived from proteolysis of inner-membrane proteins into the intermembrane space. As such peptides are presented as minor histocompatibility antigens on the surface of mammalian cells, a physiological role of ABCB10 in the antigen processing can be accounted.